From lessons on the long‐term effects of the preimplantation environment on later health to a “modified ART‐DOHaD” animal model

Abstract Background At its earliest stages, mammalian embryonic development is apparently simple but vulnerable. The environment during the preimplantation period, which only lasts a couple of days, has been implicated in adult health, extending to such early stages the concept of the developmental origin of health and disease (DOHaD). Methods In this review, we first provide a brief history of assisted reproductive technology (ART) focusing on in vitro culture and its outcomes during subsequent development mainly in mice and humans. Further, we introduce the “MEM mouse,” a novel type 2 diabetes mouse model generated by in vitro culture of preimplantation embryos in alpha minimum essential medium (αMEM). Main findings The association between ART and its long‐term effects has been carefully examined for its application in human infertility treatment. The “MEM mouse” develops steatohepatitis and kidney disease with diabetes into adulthood. Conclusion The close association between the environment of preimplantation and health in postnatal life is being clarified. The approach by which severe mouse phenotypes are successfully induced by manipulating the environment of preimplantation embryos could provide new chronic disease animal models, which we call “modified ART‐DOHaD” animal models. This will also offer insights into the mechanisms underlying their long‐term effects.

development of the fertilized embryo. 3 In mammals, fertilization normally takes place within the ampullary region of the fallopian tube, followed by preimplantation development (Figure 1). The success in generating offspring after in vitro fertilization (IVF), in vitro culture (IVC), and embryo transfer (ET) in mammals in the early to mid-20th century, allowed to treat several cases of infertility in humans. 4 Over 8 million IVF babies have been born in the world since the birth of the first IVF baby, Louise Brown, reported in 1978 by Robert Edwards and colleagues. 4 In the last decade, a number of new approaches besides IVF have been developed and integrated into routine assisted reproductive technology (ART) practices, including blastocyst stage ET, cryopreservation of embryos, and preimplantation genetic screening. 5 Although these approaches are considered beneficial for infertility treatment, vulnerable embryos unexpectedly have to experience in vitro environments that differ from those encountered in vivo ( Figure 1). The embryonic exposure to different environmental factors such as nutrition may lead to long-term consequences including altered growth and phenotype characteristics. 6 Epidemiology has studied the long-term effects of the environment in early life on the future health of individuals since the early 20th century. 7 For example, in 1934, an association between childhood conditions and later mortality was suggested from death rates for England and Wales since 1845, and for Sweden since 1751. 8 Cohort studies including one on Dutch famine near the end of World War II (1944)(1945), further revealed how extreme nutritional environments can affect fetal development and future health, leading to schizophrenia, depression, coronary heart disease, type 2 diabetes, among other disease conditions. 9,10 These studies suggest that the effects of the environment depend on their timing during gestation, with early gestation being the most vulnerable period. 9 In the last decades, these associations have been refined through various studies in a variety of research fields including clinical, epidemiological, and animal experimental research, resulting in the concept of developmental origins of health and disease (DOHaD). 11,12 According to this DOHaD concept, "the risk of developing some chronic noncommunicable diseases in adulthood is influenced not only by genetic and adult lifestyle factors but also by environmental factors acting in early life." Further, this association is expanded to refer not only to environmental exposures taking place in early life but also before life, such as those affecting the parents. 13 Thus, the concept can provide a universal platform to study the associations between environmental factors at any stage of life and the outcomes on future health.
The DOHaD concept is applicable not only to in vivo environmental factors such as the nutrition status of pregnant mothers but also to the in vitro environment of embryos notably during preimplantation, which leads to concerns regarding the effect of ART on embryos' future health. 6 In this review, we first focus on the outcomes of IVC on subsequent development and phenotypes mainly in the mouse. Second, we introduce a new unique type 2 diabetes model mouse, the "MEM mouse," which presents complications that include steatohepatitis, glomerulosclerosis, and arteriolosclerosis in the kidney as diabetic kidney disease (DKD), simply by exposure to alpha minimum essential medium (αMEM) for 48 h from the two-cell embryo stage. 14-16

| Effect of in vitro culture media on preimplantation development
In the mid-20th century, Whitten succeeded in culturing mouse embryos from the eight-cell to the blastocyst stage using a modified Krebs-Ringer-bicarbonate medium with glucose and egg white. 17 McLaren and Biggers reported a live birth after transferring embryos to the recipient uteri even after in vitro culture. 18 F I G U R E 1 Schematic flow illustrating the human in vivo and in vitro fertilization (ART). In ART, embryos experience different environments in vitro In 1959, Chang first succeeded in obtaining a live birth by rabbit IVF 19 following the finding of sperm capacitation. 20,21 About 10 years later, mouse IVF was successfully achieved. 22 Thus, over half a century has passed since the early success of in vitro embryo culture in mammals, during which, culture media have been much improved. 23,24 Two major approaches allowed to optimize their chemical composition and concentration: "back-to-nature" which aims to mimic human oviduct and uterine fluids in the female reproductive tract, resulting in the human tubal fluid medium, 25 and "let the embryos choose" which aims to maximize the developmental rate and notably yielded the KSOM medium. 26 However, even these well-developed media are not optimal and cause stress to the embryos compared to the in vivo situation. 27 Preimplantation embryos must adapt to their cultural environment to survive and, consequently in vitro culture itself impacts not only on their intrinsic developmental genetic program and viability but also on their future health. 6,13

| Impact of IVF/IVC on subsequent development and health
The numerous studies using human ART and animal models suggest that preimplantation embryos are highly vulnerable and sensitive to environmental conditions that can affect their future growth and health. 6,13 For example, poor maternal nutrition even exclusively during preimplantation development results in adult excess growth and hypertension especially in female mouse offspring. 28 After IVF compared to natural mating, the mouse offspring weigh more at birth, while females show delayed glucose clearance with more insulin secretion. 29 Therefore, human ART raised concerns in terms of increasing the risk of developing type 2 diabetes and cardiovascular diseases in adults, although more studies are needed to reach strong conclusions. 30 How can the environmental conditions of preimplantation embryos contribute, in a couple of days, to increasing such disease risk in the future? There are several good models including maternal low protein diet and IVF which allow dissecting this association. Here, we focus on the differences between IVF/IVC and in vivo embryos to provide such insights. Based on animal model studies, IVF/IVC reduces during preimplantation the number of trophectoderm (TE) cells, which give rise to tissues in the placenta, but also increases cell death of the blas-  55 Investigating how rDNA transcription is affected in embryos and adults after IVF or IVC is thus considered important. 56 While further studies identifying the genes causing the long-term effects of IVF/IVC are warranted, DNA methylation is assumed to represent one of the important changes leading to adverse developmental programming.

| Perinatal and long-term outcomes associated with IVF/IVC
Although most IVF children are healthy, accumulating evidence suggest increased risks of outcomes associated with IVF, such as stillbirth, fetal growth restriction, low birth weight, preterm birth, preeclampsia, placenta previa/accreta, increased growth trajectory in infancy, as well as metabolic and cardiovascular defects in later life, in addition to imprinting disorders as mentioned above (Figure 2). [57][58][59][60][61] An association between birth weight and later chronic diseases including cardiovascular diseases has been suggested from epidemiological observations, contributing to the DOHaD concept. 62,63 Therefore, in both IVF-and spontaneously conceived children, it is important to identify the causal mechanism underlying altered prenatal development in terms of outcomes on future health. How can we dissect causal relationships following IVF/IVC?
The theory of "placenta-derived diseases" 64 provides key insights and a comprehensive understanding of the abnormalities induced by ART, including IVF/IVC. 65 The placenta forms an interface between the fetus and its mother to sustain fetal development by providing the mother with all the nutrients and oxygen, functioning as a barrier against maternal hormones and immune system as well as parasites, and acting as an endocrine organ. 66,67 The theory of "placenta-derived diseases" stipulates that "if normal placenta is impaired or the organ's capacity for adaptation exceeded, then the fetal milieu may be perturbed with major consequences for the lifelong health of the offspring," 65 based on accumulating evidence of strong associations between placental phenotypes and chronic diseases, following the DOHaD concept. 68 Accumulating evidence suggest that ART increases the risk of abnormal placental phenotypes such as placenta previa, greater placental weight, placental metabolic alterations, and abnormal gene expression. 65 Consistently, in mouse, ART treatments reduce fetal weight and induce placental overgrowth at embryonic day 18.5, resulting in defects of placental layer segregation and glycogen cell migration. 69 These ART treatments also downregulate placental nutrient transporters and reduce placental efficiency. 69 The ART placentae exhibit increased methylation levels at ICRs of H19 with abnormal expression of imprinted genes which are important for placental development and function. 69 Another recent mouse study dissected the effect of distinct ART approaches such as hormone stimulation, IVF, IVC, and ET, which revealed that IVC itself causes placental overgrowth, as well as reduces fetal weight and placental DNA methylation, while placental expression levels of sFLT1, an anti-angiogenic protein, increase after IVF/IVC as increased circulating maternal levels of sFLT1 are implicated in causing maternal symptoms of preeclampsia in humans. 70 Therefore, among the ART procedures, IVC is considered F I G U R E 2 Schematic flow illustrating the embryo's possible short-term and long-term outcomes after ART F I G U R E 3 Schematic flow of the MEM mouse as a "modified ART-DOHaD" animal model

| "MEM mouse" as a "modified ART-DOHaD" animal model
Preimplantation as ob/ob mice. 72 Second, it is important to investigate various "ART-DOHaD" animal models, produced by ART and shown to exhibit longterm effects, 73 integrated into "DOHaD" animal models produced by maternal nutritional imbalance such as under-and overnutrition. 74 Since ART in domestic animals including cattle, sheep, and horses is worldwide used, pre-and peri-natal effects have been studied to resolve ART-associated problems such as low pregnancy rates, prolonged gestation, and fetal overgrowth, also known as the large offspring syndrome (LOS). 73 As a result of studying causative factors, for example, for LOS in cattle, which presents as an aberrant development of the placenta, 75 the inclusion of serum in embryo culture medium and co-culture with oviductal cells were identified mainly to cause abnormal feto-placental development in ruminants. 73,76 Finally, it appears critical to study different types of "modified ART-DOHaD" animal models with more severe phenotypes and a higher penetrance upon embryo exposure to synthetic microen-  Figure 3). 26,80 These results imply that the "MEM mouse" can be used as a novel animal model for human diabetes. 14

| CON CLUS ION
Over the last two decades, our understanding pertaining to ART including IVC and its long-term effects has much advanced based on the DOHaD concept. This DOHaD concept is also pertinent to other fields such as evolutionary developmental biology (evo-devo) and ecological developmental biology (eco-devo), which together provide a framework for understanding when and how environmental stressors modify the phenotypes of individuals, then result in chronic diseases over the life cycle through epigenetic regulation. 83 Combined with protocols allowing to design "modified ART-DOHaD" animal models with desired phenotypes by manipulating the microenvironment during preimplantation, studies of "modified ART-DOHaD" animal models are expected to contribute not only to improving the culture medium for ART required to produce healthy offspring, to developing drugs and foods for the treatment of chronic diseases but also to decoding the underlying developmental programs. In this review, we do not cover all the work related to ARTinduced long-term consequences considering that excellent Review papers have already been published 38,65 but instead, we insist on the importance of studying "modified ART-DOHaD" animal models which are expected to contribute to elucidating the mechanisms underlying ART-induced long-term consequences on health.

ACK N OWLED G M ENTS
We gratefully acknowledge our discussions with Drs. T. Wakayama, S. Wakayama, and M. Ooga. We are also grateful to the staff and students at the Advanced Biotechnology Center, University of Yamanashi and Mr. T. Nakagawa at Kiwa Laboratory Animals Co., Ltd. The authors would additionally like to thank Enago (www.enago. jp) for English language Review.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

H U M A N A N D A N I M A L R I G HT S
Non-applicable for a Review article.